The present invention is a method to detect the presence of live non-A, non-B hepatitis viral agents in a sample, in a body fluid, such a serum, or in a biological preparation, such as a vaccine.
Posttransfusion non-A, non-B hepatitis (NANBH) is a serious illness. It has a disturbing tendency to progress to chronic hepatitis, even more frequently than posttransfusion type B hepatitis. Additionally, a non-A, non-B hepatitis infection is likely to be followed by an asymptomatic carrier state. More importantly, aplastic anemia is often preceded by hepatitis. It has been found that most cases of hepatitis that precede aplastic anemia are believed to be caused by non-A, non-B hepatitis viral agents. The mortality rate from aplastic anemia is quite high. The modes of transmission include transfusion, hemodialysis, renal transplantation, illicit self-injection, sexual contacts and a variety of other sources. Because of the dangers of the disease and its complications, there is an incentive to immunize susceptible populations against these viral agents much the same way immunization against smallpox, polio and hepatitis B has been accomplished.
In simplest terms, a vaccine for injection into an animal for immunization from a disease contains weakened or dead viruses. The injection of the weakened or dead virus into the animal causes natural antibodies to develop within the animal and ultimately results in an immunization by that animal toward that virus.
It is essential to the proper use of any vaccine that it does not contain any live virus that could cause disease. Of course, since the vaccine is actually made from a live virus which is either weakened or killed, there is a possibility that the vaccine might contain an infectious virus. Injecting an infectious virus into an animal would contribute toward the animal's contracting the particular disease and therefore would not accomplish the objective goal of immunization, the prevention of contraction of the disease.
Accordingly, a vaccine must be tested to determine whether or not any infectious virus is present. There is no known way to detect the presence of many infectious viruses, including hepatitis viruses, in a sample except for a very costly and time consuming use of primates. Essentially, the vaccine would be injected into a chimpanzee and after a period of time of approximately one year, if the chimpanzee did not develop the disease, the vaccine would be considered safe. This process is time consuming, as a sufficient period of time must go by to ensure that the disease does not surface in the animal. The method is also expensive as each animal used is expensive and cannot be used more than once.
Besides vaccines, humans receive a variety of blood products such as clotting factors that have been associated with transmitting diseases, such as viral hepatitis. A method to detect the presence of viral agents in these products would aid in ensuring the safety of these products for human use.
There is no way of detecting non-A, non-B hepatitis virus at this time, except to infect humans and chimpanzees. Approaches used in the past to identify hepatitis A and B antigens and viral particles have been applied unsuccessfully to the search for non-A, non-B hepatitis antigens. There are no immunological or nucleic acid probes for the virus. The only indirect markers for an infection are elevations of the aminotransferases and other liver enzymes (AST,ALT) and the exclusion of other known causes of viral hepatitis.
Recently, Young et al demonstrated that in blood of people who are diagnosed as having severe anemia (a disease which expresses itself by noticeable suppression of the growth and differentiation of bone marrow cells), a parvovirus was present which inhibited the growth and differentiation of certain bone marrow stem cells (CFU-E). Young et al developed an in vitro assay that detects the inhibition of growth of the stem cell when exposed to a sample containing the virus to indicate the presence of the virus. See N. S. Young et al, "Characterization of a Virus that Causes Transient Aplastic Crisis", Journal of Clinical Investigation, 73: 224-230, 1984. This parvovirus is not associated with viral hepatitis.
More recently in pending U.S. patent application Ser. No. 893,678, filed Aug. 6, 1986, entitled "Assay to Detect the Presence of Live Virus In Vitro", a method for detecting the presence of hepatitis viruses, particularly hepatitis B virus (HBV), in a sample of body fluid or in a biological preparation was disclosed. That method involved exposing mononuclear cells, derived from bone marrow or blood, in vitro to a sample to be tested for the live virus. The cells were then suspended in a semi-solid media in the presence of growth factors that promote the proliferation and differentiation of hematopoietic stem cells. After a period of time, the stem cells form a clump of cells called a colony. Depending upon the growth factors added to the cells, different types of bone marrow stem cell colonies can be detected. The number of colonies detected after exposure to hepatitis viruses is less than those present in cultures of bone marrow cells exposed to a sample that lacks the virus.
The application of these specific serological tests for the diagnosis of infection caused by hepatitis A virus (HAV) and HBV has led to the conclusion that significant numbers of cases of acute and chronic hepatitis in humans occur in the absence of infection with any known serologically identifiable virus. These cases have been tentatively designated non-A, non-B (NANB) hepatitis to indicate that the diagnosis is based on the exclusion of infection caused by HAV or HBV. The term non-A, non-B, rather than hepatitis C, was chosen to reflect the likelihood that more than one etiologic agent may ultimately be identified. Efforts to identify specific virus particles and virus antigens have been unsuccessful. In the absence of a virus-specific test for NANB hepatitis, as well as the absence of diagnostic clinical features, the diagnosis is made not only by the exclusion of HBV and HAV but also by the exclusion of Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Additionally, the assays disclosed in U.S. patent application Ser. No. 893,678 (discussed above) were not successful in detecting the presence of non-A, non-B hepatitis virus in vitro; sera from chimpanzee or people infected with non-A, non-B hepatitis failed to inhibit the growth and differentiation of bone marrow stem cells. Based upon findings with HBV and subsequent data which is the subject matter of this application, the applicants have determined that the failure of NANB hepatitis virus to inhibit stem cells was caused by low titre of the virus and not a property of the virus as previously thought.
Accordingly, it is an object of the present invention to provide an assay to detect the presence of live non-A, non-B hepatitis virus in vitro.
It is another object of the present invention to provide an economic and accurate assay to detect the presence of live non-A, non-B hepatitis virus in vitro.
Still another object of the present invention is to provide an assay to detect the presence of live non-A, non-B hepatitis virus in a vaccine.
It is another object of the present invention to provide an assay for the in vitro screening of drugs and antibodies for the treatment of non-A, non-B hepatitis virus.